Notably, d,1-5-methyltetrahydrofolic acid is commonly prepared from folic acid by a process comprising the following essential Steps: ##STR3##
The Step (a) has been carried out both by catalytic hydrogenation and by chemical reduction, in particular by using alkaline borohydrides. (Y. Hetefi et al. - Biochem. Prep. 7, 89 (1960); K. G. Serimgeour et al. - Biochemistry 5, 1438 (1966)). In this Step, no matter what known method is used, a mixture of folic acid, tetrahydrofolic acid and dihydrofolic acid is obtained, consisting mainly of the first two components and difficult to resolve. The only useful methods comprise carrying out Step (b) with very large chromatography columns and using a series of eluents (L. Jaenicke et al. - Z.physio-. Chem. 326, 168 (1961); J. C. Keresztesy et al. - Biochem. Biophysic Res. Commun. 5, 286 (1961); H. Rudiger et al. FE/BS/Letters 4, 316 (1969)). The excess of borohydride in the solution subjected to chromatographic separation is first destroyed by an acid, preferably hydrochloric acid or acetic acid.
The methylation of the tetrahydrofolic acid (Step c) may also be carried out by various methods, the most common and convenient of which comprises reaction with formaldehyde and then reduction with a chemical reducing agent, preferably again an alkaline borohydride (W. Sakami et al., Biochem. Prep. 10, 103 (1963)).
The separation and final purification of the 5-methyltetrahydrofolic acid (Step d) has always been carried out up to the present time by chromatography processes of a greater or lesser complexity, as determined by the nature of the mixture to be purified.
In one modification of said method (J. A. Blair et al., Analytical Biochemistry 34, 376-381 (1970)), Step (b) is dispensed with and the reaction mixture in which the excess borohydride has been destroyed is treated directly with formaldehyde and another alkaline borohydride. Evidently such a modification does not substantially improve the process, because as Step (a) in any case leads to the production of a mixture as heretofore specified, dispensing with Step (b) gives a final mixture which is even more complex and difficult to resolve, with consequent aggravation of the purification Step (d), and a loss in this Step of about 60% of the product.
Furthermore, a high purity product is not obtained as would be required for use in the pharmaceutical field.
In conclusion, said process, which is the best known at the present time, has never gone beyond laboratory use and the production of small quantities of substance for experimental use.